R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

Abstract

SUMMARY R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations

“…In HEK293T cells, R-ChIP captured R-loops with the expected strand specificity that predominantly occurred at gene promoters containing highly G/C-skewed sequences, as characterized earlier (Figure S3A; Chen et al, 2017; Ginno et al, 2012, 2013). Because Dox induced both the mRNA and protein of exogenous genes at 5 hr (Figures S3B and S3C), we chose this early time point to apply R-ChIP to cells carrying SRSF2 (P95H), U2AF35 (Q157P), and U2AF35 (S34F) mutations to minimize potential indirect effects (Figure S3D).…”

“…To investigate this puzzle, we took advantage of a strategy recently developed in our lab (called R-ChIP, illustrated in Figure 3A) to map R-loops genome-wide by using the catalytically inactive RNase H1, which dramatically increases the resolution relative to that achieved with the existing strategies based on the use of the S9.6 antibody to recognize RNA:DNA hybrids (Chen et al, 2017; Ginno et al, 2012). In HEK293T cells, R-ChIP captured R-loops with the expected strand specificity that predominantly occurred at gene promoters containing highly G/C-skewed sequences, as characterized earlier (Figure S3A; Chen et al, 2017; Ginno et al, 2012, 2013).…”

“…A comparison of induced R-loops in three mutant cells showed limited overlap (Figure S3H), suggesting that different mutations may preferentially induce R-loops in different sets of genes. Alternatively, such a limited overlap might be due to the highly dynamic nature of R-loop formation and resolution (Chen et al, 2017), of which we were only able to capture a snapshot in each profiling experiment.…”

“…R-ChIP was developed to capture R-loop in cells expressing a V5-tagged RNASEH1 (D210N) mutant gene (Chen et al, 2017). The protocol follows a standard ChIP strategy with modifications (Ji et al, 2013).…”

What viruses tell us about evolution and immunity: beyond Darwin?

“…Small RNA‐mediated paramutations (see below) can last for at least 20 generations in the nematode C. elegans . Recently, tRNAs and R‐loop formation have also been found as mechanisms that contribute to epigenetic changes of gene expression …”